Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Abstract

Active demethylation of 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER). The TDG-BER pathway may lead to the generation of DNA strand breaks, potentially compromising genome integrity. Alternatively, direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks. However, cleavage of the C-C bond in 5caC in human cells remains an open question. We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate. After incubation with whole-cell protein extract or transfection into human cells, we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at both the mononucleotide and oligodeoxynucleotide levels. Our results clearly showed the direct conversion of 5caC to cytosine in human cells, providing evidence to support a novel pathway for active DNA demethylation.

DNA methylation modification is associated with gonadal differentiation in Monopterus albus

“…The liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) were performed using the method described previously with slight modification [ 22 , 24 ]. The LC–ESI–MS/MS system consisted of a Shimadzu LC-20AD high performance liquid chromatography (HPLC) (Tokyo, Japan) and an AB 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA) with an ESI source (Turbo Ionspray).…”

Novel dual methylation of cytidines in the RNA of mammals

“…DNA cytosine methylation (5-methylcytosine, 5 mC) is the most important epigenetic modication that can regulate gene expression in genomes of higher eukaryotes. [1][2][3] Similar to the 5 mC on DNA, RNA modications recently were also discovered to have critical roles in regulating a variety of physiological processes. 4,5 Up to date, over 150 structurally distinct modied nucleosides have been identied in various RNA species in all three kingdoms of life.…”

“…6 m 3 C was initially discovered in yeast RNA and later on was identied to be present in both the rRNA and tRNA of eukaryotes. 7 Recently, m 3 C has also been found in the mRNA of mammals. m 3 C carries positive charges and could affect the secondary structure of RNA, change protein-RNA interactions and affect translation.…”

“…23 Although m 4 Cm was identied to be present in the 16S rRNA of E. coli, it hasn't been reported in the RNA of mammals. Moreover, m 3 Cm, m 4 Cm and m 5 Cm are structurally isomers and have exactly the same molecular weights, which causes the distinct determination of these dimethylated cytidines a challenging task. Some methods have been developed for determining RNA modications, such as two-dimensional cellulose thin layer chromatography (2D-TLC), 24 liquid chromatography (LC) coupled with ultraviolet 25 or mass spectrometry (MS) detection.…”

“…Because of the high detection sensitivity and good capability for deciphering structures, LC-MS recently has become a more widely used platform for analyzing lowabundant nucleic acid modications. 26,33,34 In the current study, we established a liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/ MS) method to determine the dimethylation of cytidines, m 3 Cm, m 4 Cm and m 5 Cm. Under optimized analytical conditions, m 3 Cm, m 4 Cm and m 5 Cm can be clearly distinguished.…”